The objective of this exploratory (R21) proposal is to identify the most promising strategy for solving the contrast problem in electron microscopy. In all microscopes, whether illuminated by photons or electrons, biological tissues are essentially transparent. Green Fluorescent Protein (GFP) and related fluorescent proteins have complemented small molecule stains to essentially solve the contrast problem in optical microscopy. For electron microscopy, however, there are no 'clonable' contrast markers analogous to GFP. Herein we propose to explore several peptide or small protein based strategies for nucleating, catalyzing or mediating the formation of electron dense inorganic nanoparticles which may allow the localization of individual proteins in macromolecular and whole cell electron microscopy. The proposed work is significant for four areas of inquiry. First and most significantly, a robust clonable nanoparticle will revolutionize electron microscopy, making it significant for all forms of biological EM. Second, we will study FtsZ, the prokaryotic tubulin analog as an initial target. Information we learn about the intrcellular structure and distribution of FtsZ is significant for the development of antibiotics that target FtsZ. Third, we will, in finding novel peptides or small proteins that interact with metal ions or coordination complexes learn more about the important and sometimes poorly understood interactions of metals and biomolecules. Fourth just as GFP can act as a 'reporter gene' for reading optically for gene expression in tissues and organisms, a genetically encodable magnetic nanoparticle may also serve as a widely used 'reporter gene' for detecting gene expression by MRI or CT. The proposed work will proceed in three specific aims. All candidate 'clonable nanoparticle' peptides or proteins identified in these specific aims will be evaluated in vitro, in situ and in vvo in an FtsZ based structural electron microscopy assay. Aims one and two use the directed evolution techniques of phage display and ribosome display, respectively, to isolate peptide sequences capable of provoking the formation of magnetic iron oxide nanoparticles. Aim 2 is technically more challenging but is more likely to yield a universally useful clonable nanoparticle than Aim 1, which may yield clonable nanoparticles that function in situ and in vitro, but not in vivo. Aim 3 is to assay naturally occurring peptides, proteins and relevant protein fragments, as well as peptides isolated by others, for comparison to the peptides we isolate in aims 1 and 2. The proposal identifies why existing 'known' peptides and proteins fail to function as EM contrast markers, and suggests modifications that we may make to these known peptides and proteins to adapt them as EM contrast markers. The public health significance of the development of the proposed enabling technologies will derive from both more comprehensive structural information on normal and diseased cells, and also from greater understanding of FtsZ biology, which may enable development of new antibiotics. PUBLIC HEALTH RELEVANCE: Microscopes are critical to the diagnosis and understanding of disease. Biological material is essentially transparent in both light and electron microscopes. This 'contrast problem' was essentially solved for light microscopy by fluorescent proteins, as recognized by the 2008 Nobel prize in chemistry. Inspired by fluorescent proteins, we propose to implement a strategy of clonable nanoparticles for solving the contrast problem in the much higher resolution technique of electron microscopy.